Xeno-free generation of tissue-specific progenitor cells

ABSTRACT

The invention relates to purified, tissue-specific progenitors, methods of making and using such tissue-specific progenitors.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of US Provisional Patent ApplicationNo. 61/382,095, filed Sep. 13, 2010, incorporated herein by reference asif set forth in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under HL081076 awardedby the National Institutes of Health. The government has certain rightsin this invention.

BACKGROUND

Tissue-specific progenitor cells, also known as tissue-specific or adultstem cells, are rare populations of cells present in many adult tissuescapable of differentiating into various cells specific to the tissue inwhich they reside. For example, hematopoietic stem cells (HSCs) are arare population of cells inside the bone marrow that are responsible forgenerating all types of blood cells. Similar tissue-specific progenitorcells reside in other tissues, such as brain, heart, liver, and pancreasand can give rise to cells of their respective tissues. These cells holdgreat promise for clinical use to regenerate damaged or lost tissue.Clinical use has been hampered, in part, by an inability to isolate orproduce sufficient numbers of tissue-specific progenitor cells suitablefor clinical application. At present, HSCs are the only adult stem cellsin clinical use, but their use is restricted by the limited availabilityof these cells. There is, thus, a need in the art for methods ofproducing tissue-specific progenitor cells in vitro that are suitablefor clinical application.

Hematopoietic stem cells (HSCs) are the best-characterized example oftissue-specific progenitor cells. Successful engraftment of a smallnumber of CD34⁺ HSCs can sustain hematopoiesis for a lifetime. The studyof human hematopoiesis has been greatly advanced by the development ofmethods to generate HSCs from human embryonic stem cells (hESCs)(Kaufman et al., PNAS 98(19):10716 (2001); Vodyanik et al., Blood105(2):617(2005)). Effective methods of generating tissue-specificprogenitor cells suitable for clinical use, such as HSCs, from hESCscould provide a novel source of progenitors for transplantation. Inaddition, hESC-derived tissue-specific progenitor cells can be used toproduce various tissue cells that can be used for clinical andpharmaceutical research or can be administered to individuals in needthereof.

Unfortunately, currently available methods include culturing hESCs onmurine bone marrow stromal cells, which is undesirable for preparingcells intended for clinical use (Nakano et al., Science 265(5175):1098(1994)). Within the body, HSCs are maintained in an undifferentiatedstate within bone marrow microenvironments or “niches.” These HSC nichesare thought to regulate survival, self-renewal, and maintenance of HSCsthrough growth factor and cytokine secretion, structural support, anddirect cell-to-cell crosstalk. The cellular microenvironment iscomprised of various cell types in the bone marrow stromal includingmesenchymal stem cells (MSCs), vascular endothelial cells, and reticularcells.

Mesenchymal stem cells (MSCs) are fibroblast-like cells that residewithin virtually all tissues and organs of a postnatal individual andcan differentiate into cells of the mesenchymal lineage, such asosteoblasts, adipocytes, and chondrocytes. MSCs have been isolated frombone marrow, adipose tissue, heart, pancreas, liver, kidney, and othertissues. Tarnok et al., Cytometry 77(1):6-10 (2010). Within the bonemarrow niche, MSCs support survival, proliferation, and differentiationof HSCs and their progeny through a variety of mechanisms, such as byproducing extracellular matrix components for structural support and bysecreting growth factors and cytokines that support HSC maintenance andproliferation. Long-term bone marrow cultures demonstrated theimportance of MSCs in hematopoietic stem and progenitor cell maintenanceex vivo and MSCs have provided invaluable tools for investigating thestem cell niche in both normal and malignant hematopoiesis. Human MSCscan support hESC expansion in vitro (Cheng et al., Stem Cells 21:131(2003)).

Apart from MSCs, a wide variety of other cell types contribute to normalbone marrow function. For example, osteoblasts, which are differentiatedprogeny of MSCs, are critical in HSC niche maintenance while adipocytes,also differentiated progeny of MSCs, are negative regulators ofhematopoiesis.

Macrophages are present in almost all tissues and are essential toinnate immunity. Like other hematopoietic cells, macrophages originatefrom a bone marrow progenitor cell that first gives rise to monocytes.Monocytes circulate in the peripheral blood and can give rise tomacrophages after extravasating from the blood stream into thesurrounding tissue, either to replace long-lived tissue macrophages orin response to injury. Gordon, European J Immunol. 37 Suppl 1:S9-17(2007). Within the tissue, macrophages can be polarized by theirmicroenvironment to assume different phenotypes. Stout et al., J.Immunol. 175:342-349 (2005). For example, certain macrophages areessential for all stages of tissue repair including chemotaxis, matrixremodeling, epithelial migration, and angiogenesis (Pollard, Nature Rev.9:259-270 (2009)).

The data on macrophage involvement in hematopoiesis are conflicting.Macrophages have been implicated in erythropoiesis. Transmissionelectron micrographs showed erythroblasts surrounding a centralmacrophage. Bessis and Breton-Gorius, Blood 19:635-663 (1962). These“erythroblastic islands” play a crucial role in normal erythroiddevelopment by providing iron for heme synthesis and erythropoietin forerythropoiesis to developing erythroblasts. Abnormal erythroblasticislands are associated with altered erythropoiesis of pathologicalprocesses such as anemia of inflammation and chronic disease,myelodysplasia, and thalassemias. Chasis et al., Blood. 112:470-478(2008). In all these conditions, the role of macrophages has beenassumed to be restricted to erythropoiesis.

In contrast, recent studies suggest that monocytes and macrophagesnegatively affect in vitro expansion of HSC and hematopoiesis (Yang etal., Bone Marrow Transplant. 45(6):1000 (2010); Jaiswal et al., Cell138:271 (2009)). A recent study suggests that HSCs respond toinflammatory stimuli and upregulate CD47, which then interacts withmacrophage receptors to evade macrophage-mediated destruction among thetoxic inflammatory milieu. Thus, the role of macrophages as adirect-acting component of the HSC niche was unknown prior to theinventors' work. Further, it was not known whether MSCs and macrophagesinteract and whether such interactions affect survival and proliferationof HSCs.

While HSCs have been studied extensively, little is known abouttissue-specific progenitor cells of other tissues. Until recently, itwas believed that the heart and brain contained only terminallydifferentiated cells unable to proliferate. However, recent studiesidentified a subpopulation of cells in the heart, brain, and otherorgans that are able to proliferate and repopulate damaged or destroyedtissues. There is a great need in the art for methods for enhancingproliferation of these cells in vivo or in vitro.

Interactions between macrophages and tumor cells in hematologicalmalignancies, with the exception of follicular lymphoma, are not wellunderstood. Recent studies suggest that macrophages can promoteangiogenesis in multiple myeloma (MM), the second most commonlydiagnosed hematological malignancy in the developed world. Also,macrophages might protect myeloma cells from spontaneous andchemotherapy-induced apoptosis. Zheng et al., Blood 22;114(17):3625-3628 (2009). However, the role of BM macrophages as adirect-acting component in the MM tumor niche has not been recognized.Further, it has not been investigated if MSCs-macrophage interactionaffects survival and proliferation of MM cells. The multitude ofcellular compartments and the broad constellation of growth factors andcytokines involved in the MM tumor niche pose significant therapeuticchallenges. Targeting any individual molecular or cell mediator of theMM BM milieu is not sufficient for curative responses due to functionalredundancy supporting MM cell survival. New models to investigate thefunctional hierarchy of the BM microenvironment are necessary to deviseeffective therapeutic strategies.

Prior to the inventors' work, it was unknown whether cellularinteraction of MSCs with another cell type can affect function of athird cell type. Prior to the inventors' work, it was further unknownwhether the origin of the MSC affects the quality of interaction with,and subsequent fate of, another cell.

BRIEF SUMMARY

The present invention is broadly summarized as relating to methods forgenerating tissue-specific progenitor cells from pluripotent cells.While progenitors have been generated in culture, they required culturewith cells from different species, e.g., human pluripotent cells onmurine feeder cells. The inventors developed novel methods that employcells of only one species.

In a first aspect, the invention is summarized in that a method forproducing a tissue-specific progenitor includes the step of co-culturinga CD14⁺ cell, a tissue-specific MSC, and a pluripotent cell in vitro toproduce the tissue-specific progenitor cell. In some embodiments of thefirst aspect, all three cells are from the same species. In someembodiments of the first aspect, the method is conducted undercompletely xeno-free conditions.

In a second aspect, the invention relates to a population oftissue-specific progenitor cells generated by co-culturing a CD14⁺ cell,a tissue-specific MSC, and a pluripotent cell in vitro to produce atissue-specific progenitor cell. In some embodiments of the secondaspect, the tissue-specific progenitor cells express CD26.

In a third aspect, the invention relates to a cell culture compositioncomprising a CD14⁺ cell, a tissue-specific MSC, and a pluripotent cell.Some or all of the cells of the cell culture composition can beallogeneic with regard to each other. Likewise, some or all of the cellsof the cell culture composition can be syngeneic or autologous to eachother.

In a fourth aspect, the invention relates to a cell culture compositioncomprising a CD14⁺ cell, a tissue-specific MSC, a pluripotent cell, anda tissue-specific progenitor cell. The composition can optionallyfurther comprise a compound of interest to investigate the compound'seffect on the culture composition.

In a fifth aspect, the invention relates to methods for treating adisorder associated with aberrant loss of normal cell functioncomprising the step of administering to an individual in need thereof atissue-specific progenitor cell. The progenitor cell used in thesemethods can be allogeneic, syngeneic, or autologous with regard to theindividual.

In a sixth aspect, the invention relates to methods for in vitro testingof compounds by co-culturing a CD14⁺ cell, a tissue-specific MSC, and apluripotent cell in vitro to produce the tissue-specific progenitor celland then contacting the progenitor cell with the compound of interest.The progenitor cell can be cultured either under conditions that promoteor prevent differentiation, depending on whether the compound is to betested for its effect on undifferentiated progenitors or thedifferentiation process. Alternatively, the compound of interest can beadded to the CD14⁺ cell, tissue-specific MSC, and pluripotent cellculture composition prior to production of the tissue-specificprogenitor cell.

In a seventh aspect, the invention relates to methods for culturing amalignant cell in vitro by co-culturing a malignant cell with a CD14⁺cell such that the malignant cell proliferates. The CD14⁺ cell can be anMSC-educated macrophage. As used herein, “MSC-educated macrophage”refers to a macrophage that was generated by co-culturing a CD14⁺ cellwith an MSC. Optionally, an MSC can be added to the malignant cell-CD14⁺cell co-culture. The malignant cell, such as a myeloma cell, can beisolated from a human individual.

In an eighth aspect, the invention relates to methods for culturing aCD34⁺ cell in vitro by co-culturing a CD34⁺ cell with a CD14⁺ cell invitro such that the CD34⁺ cell proliferates. The CD14⁺ cell can be anMSC-educated macrophage. Optionally, the CD34⁺ cell is co-cultured witha CD14⁺ cell and an MSC.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although suitable materials andmethods for the practice or testing of the present invention aredescribed below, other materials and methods similar or equivalent tothose described herein, which are well known in the art, can be used.

These and other features, objects and advantages of the presentinvention will become better understood from the description thatfollows. In the description, reference is made to the accompanyingdrawings, which form a part hereof and in which there is shown by way ofillustration, not limitation, embodiments of the invention. Thedescription of preferred embodiments is not intended to limit theinvention to cover all modifications, equivalents and alternatives.Reference should therefore be made to the claims recited herein forinterpreting the scope of the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The present invention will be better understood and features, aspectsand advantages other than those set forth above will become apparentwhen consideration is given to the following detailed descriptionthereof. Such detailed description makes reference to the followingdrawings, wherein:

FIG. 1 illustrates one embodiment of a possible co-culture protocol fordeveloping tissue-specific progenitor cells.

FIG. 2A-D illustrate results of flow cytometric analysis. FIG. 1A showsthat co-cultures of ESC, bone marrow-derived MSC, and CD14⁺ cellsproduce CD34⁺ cells. FIG. 1B shows that co-cultures of ESC, pancreaticislet-derived MSC, and CD14⁺ cells produce very few CD34⁺ cells. ESC andCD14⁺ cells used were the same as in FIG. 1A. FIG. 1C shows that ESC-MSCco-cultures without CD14⁺ cells did not lead to generation of CD34⁺cells. FIG. 1D shows that the addition of CD14⁺ cells to ESC-MSCco-cultures results in generation of CD34⁺ cells. The same ESCs and bonemarrow MSCs were used for FIGS. 1C and 1D.

FIG. 3A-D illustrate animal engraftment of CD34⁺ cells produced by thedescribed co-culture protocol. Peripheral blood cells from control (FIG.3A, FIG. 3B) or engrafted (FIG. 3C, FIG. 3D) sheep were analyzed by flowcytometry using isotype control antibodies (FIG. 3A, FIG. 3C) oranti-CD45 antibody (FIG. 3B, FIG. 3D).

FIG. 4 illustrates an example of CD 34⁺ cell yield after bone marrowMSCs were co-cultured with ESCs and macrophages. The population wasgated to exclude macrophages and apoptotic cells.

FIG. 5 illustrates an example of in vitro screening for compounds thatmodulate tissue-specific progenitor cell development. Bone marrow MSCswere co-cultured with ESCs and macrophages in the presence (Wnt pathwayagonist) or absence (control) of the Wnt pathway agonist2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidineand analyzed by flow cytometry.

FIG. 6 illustrates proliferation indices of U266 and NCI-H929 MM celllines.

FIG. 7 illustrates the percent apoptotic chronic lymphocytic leukemiacells in culture either alone (CLL alone), in the presence ofmacrophages (MQ), MSCs (MSC), or macrophages and MSCs (MSC+MQ).

FIG. 8 illustrates normalized apoptotic ratios for primary B-cellscultured alone (control), or in the presence of macrophages (MQ), MSCs(MSC), or macrophages and MSCs (MSC MQ).

FIG. 9 A-E illustrate flow cytometric analysis of CD 34⁺ cells producedby co-culture of bone marrow MSCs with ESCs and macrophages.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention broadly relates to methods for generation oftissue-specific progenitor cells from pluripotent cells. Theseprogenitor cells can be generated by co-culturing tissue-specific MSCswith pluripotent cells and CD14⁺ cells. The novel method is useful forproducing adult stem cells/progenitors of desired tissue lineages forresearch and clinical application. Advantageously, the novel method canbe conducted under xeno-free conditions.

As used herein, “xeno-free” or “xeno-free conditions” refers to cellularco-culture using only cells from a single species, i.e., the cells ofthe co-culture are allogeneic, syngeneic, or autologous with respect toeach other.

As used herein, “monocyte” refers to a mononuclear leukocyte that candifferentiate into a macrophage.

As used herein, “macrophage” refers to a mononuclear phagocytecharacterized by the expression of CD14 and lack of expression ofdendritic cell markers.

As used herein, “CD14⁺ cell” refers to a monocyte or a macrophage.

CD14⁺ cells and pluripotent cells used for co-culture can be obtainedfrom any suitable source. The skilled artisan will appreciate theadvantageous efficiency of generating macrophages from peripheral bloodmonocytes for co-cultures. Alternatively, macrophages can also beisolated from individuals directly, such as through cellular outgrowthfrom tissue samples. Likewise, the skilled artisan will appreciate theadvantageous efficiency of using induced pluripotent stem cells,especially those from autologous somatic cells, as pluripotent cells.Alternatively, pluripotent cells can be obtained from various sources,such as commercially available stem cell lines.

Likewise, MSCs used for co-culture can be obtained from any suitablesource. The source of the MSC directs differentiation of the pluripotentcells in co-culture. For example, MSCs from bone marrow directpluripotent cell differentiation towards the hematopoietic lineage. MSCsfrom the heart direct pluripotent cell differentiation towards thecardiac lineage.

As such, selecting a source of the MSC can depend upon the desiredculture objective. For example, if the culture objective is to generatea hematopoietic stem cell, then MSCs from bone marrow are appropriate.Likewise, if the desired progenitor cell is a cardiomyocyte progenitorcell, then heart tissue can be an appropriate choice for MSCs. MSCs canbe isolated from virtually every tissue and organ including, but notlimited to, bone marrow, pancreas, heart, adipose tissue, lung, liver,skin, kidney, and thyroid gland. MSCs can also be produced frompluripotent cells, such as embryonic stem cells and induced pluripotentcells. Trivedi and Hematti, Exp. Hematol. 36(3):350-359 (2008).

FIG. 1 illustrates one possible co-culture protocol. According to theillustrated embodiment, MSCs derived from a desired tissue are platedinto a culture dish and allowed to adhere, e.g., for approximately 24hours. MSCs attach well to plastic surfaces but can be grown on anysuitable surface (e.g., glass, ceramic, polymers) or matrix (e.g.,collagen, laminin, MATRIGEL™). Pluripotent cells, such as embryonic stemcells or induced pluripotent stem cells, can then be added to the MSClayer and, optionally, cultured for expansion. Alternatively, MSCs andpluripotent stem cells can be added together. CD14⁺ cells are then addedto the MSC-pluripotent cell culture. In some embodiments, all three celltypes are added to the culture together. In some embodiments, thepluripotent stem cells or macrophages are placed in culture first,followed by the addition of the remaining two cell types (together or insequence). In some embodiments, alpha 20 medium (alpha-Modified EagleMedium, 20% FBS) is used for culture. Other media can be used for one ormore of the culture steps, such as R10 medium (RPMI1640 medium, 10%FBS), MSC medium (alpha-Modified Eagle Medium, 20% FBS), and C10 medium(CMRL1066 medium, 10% FBS). In some embodiments, more than one kind ofmedium can be used. For example, TeSR medium can be used for aMSC-pluripotent cell culture step, followed by a MSC-CD14⁺cell-pluripotent cell co-culture in alpha 20 medium.

The co-culture can be maintained under normoxic (21% oxygen) or hypoxic(5% oxygen) culture conditions. Incubation time required for tissuespecific progenitor cell generation can vary depending on the cultureobjective. Fresh media are typically provided every two to three days,for example, by removing half of the spent media and adding fresh mediato the culture to restore the desired culture volume. In someembodiments, monothioglycerol is added to the culture (e.g., at 300 μM)to prevent oxidative damage. Co-cultures can subsequently be tested forthe presence of progenitor cells by assessing, for example, cellmorphology or cell marker expression.

MSCs, CD14⁺ cells, and pluripotent cells can be autologous, syngeneic,or allogeneic with respect to each other. The skilled artisan willappreciate the advantageous efficiency of generating progenitor cellsfrom pluripotent cells that were derived from the patient who is toreceive the progenitor cells. For example, somatic cells, such as skincells could be reprogrammed to pluripotent cells. These cells aregenetically identical to the patient and, as such, the resultingprogenitor cells produced by Applicants' methods are also geneticallyidentical to the patient, reducing the risk of adverse reaction afteradministration.

Monocytes isolated from peripheral blood can be cultured for varioustimes and under various conditions before addition to the MSC-ESCculture or can be added directly. The skilled artisan will appreciatethat monocytes/macrophages, pluripotent cells, and MSCs employed inmethods described herein can be cultured in any medium that supportstheir survival and growth. Further, co-cultures do not require theaddition of cytokines, although cytokines or growth factors can beadded.

Monocytes or macrophages can be co-cultured with MSCs and pluripotentcells such that the cells are in direct physical contact. Alternatively,the monocytes or macrophages can be placed in a subcompartment separatefrom the subcompartment containing the MSCs and pluripotent cells, suchthat the subcompartments are in fluid communication but separated by asemi-permeable membrane. The semi-permeable membrane allows the exchangeof soluble media components and factors secreted by the cells but isimpenetrable to the cells themselves. The pores within thesemi-permeable membrane typically are between 0.1-1.0 μm, but other poresizes can be suitable.

The co-cultured cells can be allogeneic, syngeneic, or autologous withrespect to each other. As used herein, “allogeneic” refers to cells ortissues taken from different individuals of the same species that arenot genetically identical. As used herein, “syngeneic” refers to cellsor tissues that are genetically identical or closely related. As usedherein, “autologous” refers to cells or tissues taken from the sameindividual that are genetically identical.

The resulting tissue-specific progenitor cells described herein arereadily distinguished from undifferentiated pluripotent cells in thatthey assume different morphology and/or express a unique set of markers.For example, HSCs differ from undifferentiated cells in that theyexpress CD 34. HSCs are medium sized with large nuclei eccentricallysurrounded by narrow rims of cytoplasm that appears deep blue afterGrünwald-Giemsa staining and occasionally contains cytoplasmic granules(Stella et al., Haematologica 80(4):367 (1995)).

Various methods of cell separation are known in the art and can be usedto separate the resulting tissue-specific progenitor cells from theMSCs, macrophages, and undifferentiated cells depending on factors suchas the desired purity of the isolated cell populations. Tissue-specificprogenitor cells can be maintained in culture in any medium thatsupports their in vitro growth and survival. Also, tissue-specificprogenitor cells can be stored using methods known in the art including,but not limited to, refrigeration, cryopreservation, vitrification, andimmortalization.

It is contemplated that the tissue-specific progenitor cells can beadministered to an individual for treating conditions associated withaberrant tissue maintenance, repair, or function. Conditions associatedwith aberrant tissue maintenance, repair, or function include, but arenot limited to, myocardial infarction, diabetes, aplastic anemia, heartfailure, cirrhosis, and liver failure. Specifically contemplated hereinare methods for supporting and/or restoring hematopoiesis in anindividual in need thereof, comprising administering an HSC produced bythe described methods to an individual in need thereof. The ESC-derivedHSCs can also be used to generate progenies of HSCs in vitro, such asRBC and platelets, for transfusion purposes.

It is specifically contemplated that HSC produced by the describedmethods can be administered to an individual to induce tolerance tocells and cell products that originate from the same source as theengrafted cells. For example, beta islet cells produced from an ESC linewill be tolerated by individuals that previously received HSC producedfrom the same ESC line.

It is specifically contemplated that one can provide sufficientautologous tissue-specific progenitors for clinical application byco-culturing autologous monocytes or macrophages with allogeneic MSCsand pluripotent cells from a universal source. Tissue-specificprogenitors administered to an individual can be autologous, syngeneic,or allogeneic with respect to the individual. One of skill in the artwill appreciate the advantageous efficiency of using an autologousprogenitor, i.e., the administered progenitor was derived from apluripotent cell that was taken from the same individual as therecipient.

Another application for tissue-specific progenitors contemplated by theinventors is an in vitro screening method for compounds that modulateprogenitor cells and their developmental or differentiation processes.For example, to determine how a compound affects hematopoiesis,hematopoietic progenitors, such as HSCs, can be produced by applicants'method and cultured in the presence of the compound of interest. As usedherein, “modulate” means promote, enhance, inhibit, or in any other wayalter normal cell function.

The progenitors can be administered to an individual through anysuitable delivery method. A delivery method can include topicalapplication, such as application to a wound. For example, progenitorscan be delivered in a pharmaceutically acceptable carrier or dressing,examples of which include a liquid, oil, lotion, salve, cream, foam,gel, paste, film, or hydrogel. Parenteau, Regen. Med. 4(4):601-611(2009). Exemplary carriers and dressings having suitable properties arewell-known by those of ordinary skill in the art. The choice of aspecific carrier is influenced by factors such as nature of thecondition, number of cells to be administered, route of administration,and duration of treatment. Urbaniak Hunter et al., Adv. Exp. Med. Biol.671:117-130 (2010). Progenitors can also be delivered through local orsystemic injection, by surgical transplantation or implantation, or byother methods known in the art. Progenitors can be autologous,syngeneic, or allogeneic with respect to the receiving individual andwith respect to MSCs and macrophages used for their generation.

The term “tissue-specific MSC” as used herein, means an MSC isolatedfrom a single tissue or organ of an individual.

As used herein, “tissue-specific progenitor cell” means a progenitorcell committed to a specific tissue lineage.

Another application for MSC-macrophage cocultures contemplated by theinventors is ex vivo expansion of cells isolated from an individual.These cells can include undifferentiated cells, such as multipotent andpluripotent stem cells, or abnormal cells, such as malignant cells.

The invention will be more fully understood upon consideration of thefollowing non-limiting Examples.

EXAMPLES Example 1 Isolation of Human Monocytes and Mesenchymal StemCells

All protocols were approved by the Health Sciences Institutional ReviewBoard of University of Wisconsin-Madison School of Medicine and PublicHealth.

Monocytes were derived from peripheral blood of human volunteers.Monocytes were isolated from human peripheral blood using magnetic beadseparation according to manufacturers' instructions, briefly describedas follows. Peripheral blood mononuclear cells were isolated from theblood of healthy volunteers by density gradient separation using Percoll(GE Healthcare Bio-sciences, Piscataway, N.J., USA). Contaminating redblood cells were lysed by incubating the mononuclear cells in ACK lysisbuffer for 3 minutes, followed by a wash in phosphate buffered saline(PBS). To reduce platelet contamination, the cells were centrifuged at700 rpm for 15 minutes. The resulting cell pellets were resuspended andincubated with MicroBeads conjugated to anti-human CD14 antibodies(Miltenyi Biotech, Auburn, Calif., USA) for 15 minutes at 4° C. Thecells were washed to remove unbound antibody, and separated usingautoMACS Pro Separator (Miltenyi Biotech). Ninety-five percent of thepurified cells expressed CD14, as determined by flow cytometry. PurifiedCD14⁺ monocytes were plated into the wells of 6-well cell culture platesat a concentration of 0.5-1×10⁶ cells per well in Iscove's ModifiedDulbecco's Medium (IMDM) supplemented with 10% human serum blood type AB(Mediatech, Herndon, Va., USA), 1× non-essential amino acids(NEAA-Lonza, Walkersville, Md., USA), 4 mM L-Glutamine (Invitrogen,Carlsbad, Calif., USA), 1 mM Sodium pyruvate (Mediatech), and 4 ug/mlrecombinant human insulin (Invitrogen). To generate macrophages, CD14⁺monocytes were cultured at 37° C. with 5% CO₂ for approximately 3-7days, without adding any cytokines, changing the media once 3-4 daysafter initiating the cultures.

MSCs were isolated from filters left over from standard filtration ofbone marrow harvests from healthy donors. Briefly, the bone marrow cellstrapped in the filter were collected by washing the filter with PBS.Mononuclear cells were separated using Ficoll-Hypaque 1.073 (GEHealthcare Bio-sciences) and Leucosep tube (Greiner Bio-one, Monroe,N.C., USA) according to the manufacturer's instructions. Contaminatingred blood cells were lysed by incubating the mononuclear cells in ACKlysis buffer for 3 minutes. Mononuclear cells were suspended in alphaminimum essential medium (αMEM) supplemented with 10% fetal bovine serum(FBS, Hyclone, Logan, Utah, USA), 1× NEAA, and 4 mM L-Glutamine. Cellsthat attached to the culture dish (passage 0) were harvested usingTrypLE cell dissociation enzyme (Invitrogen) and then replated into newflasks, as described by Trivedi and Hematti, Exp. Hematol. 36:350-359(2008), incorporated herein by reference as if set forth in itsentirety. Passage 4 cells were characterized by flow cytometry and bymulti-differentiation assays for MSC characteristics (Dominici et al.,Cytotherapy 8(4):315 (2006)).

Example 2 Bone Marrow-Derived MSCs and Macrophages StimulateDifferentiation of hES Cells Into CD34⁺ Cells In Vitro

Cells

Monocytes and MSCs were isolated essentially as described in Example 1.The Embryonic stem cell (ESC) line WA01 was supplied from WiCellInstitute. ESCs were maintained on Matrigel™-coated plates (BDBiosciences, San Jose, Calif.) in TeSR media (WiCell, Madison, Wis.)according to standard protocol published by WiCell Institute (Ludwig etal. Nat. Methods 3(8):637-646 (2006), incorporated herein by referenceas if set forth in its entirety). Briefly, ESCs cultured on Matrigel™were treated with 1 mg/ml dispase (Invitrogen, Carlsbad, Calif.) for 7minutes (5 minutes for xeno-free matrix cultured ESC). Cells were washedwith DMEM/F12 three times and were collected by scraping with a glasspipette. The harvested cells were gently pipetted to dissociate largecolonies and were added to Matrigel™-coated plates for expansion.

MSC-ESC Co-Culture

MSCs were plated into 6-well plates at a density of 1×10⁵-3×10⁵ cellsper well in media. The media used at this stage were determined by thetarget tissue in question but any medium containing FBS was determinedto be suitable for preparing MSC feeder layers for ESCs. For bone marrowMSC plating, R10 medium was used, which contains RPMI1640 medium(Mediatech, Manassas, Va.) supplemented with 10% FBS (AtlantaBiologicals, Lawrenceville, Ga.), 1% non-essential amino acid, 1%L-Alanine-L-Glutamine and 1% Sodium pyruvate (Mediatech, Manassas, Va.).After 1 hour, plates were ready for use as ESC feeder. ESCs wereharvested using dispase treatment as described above. ESCs weremaintained on MSC feeders for several days in TeSR media to allow forESC expansion and colony formation (about 3-4 days).

MSC-ESC-CD14⁺ Cell Co-Culture

CD14⁺ cells were added to ESC-MSC co-cultures at a concentration ofabout 1×10⁶ cells per well. The CD14⁺ cells used for these experimentswere either monocytes from peripheral blood that were fresh or storedfrozen, or macrophages derived from CD14⁺ monocytes by culturingmonocytes for more than 1 week in vitro. The ESC-MSC-CD14⁺ cellco-cultures were incubated in R10 medium, which was changed every threeto four days by collecting floating cells in the supernatant throughcentrifugation and resuspending them in fresh medium which was thenadded back to the original culture plates.

Several media and media combinations were used for MSC-ESC- andMSC-ESC-CD14⁺ cell co-culture, such as alpha 20 medium (alpha-ModifiedEagle Medium, 20% FBS) R10 medium (RPMI1640 medium, 10% FBS), MSC medium(alpha-Modified Eagle Medium, 20% FBS), and C10 medium (CMRL1066 medium,10% FBS). Other media that can support progenitors of a desired lineagecan be used with the described culture methods. Also, MSCs, pluripotentcells, and CD14⁺ cells were added to the co-culture together or invarious sequence. MSCs or CD14⁺ cells each adhered readily to untreatedculture dishes and supported subsequent pluripotent cell adhesion.

To determine the effect of low oxygen on the generation of tissuespecific progenitor cells, some of the culture experiments wereconducted under hypoxic conditions. Oxygen levels were reduced to 5%within cell culture incubators by using the nitrogen purge method,essentially as described by Simon et al, Nat. Rev. Mol. Cell Biol.9(4):285 (2008), incorporated herein by reference as if set forth in itsentirety.

ESC-MSC-Macrophage Co-Culture Analysis Using Flow Cytometry

For flow cytometric analysis, cells were collected by aspirating medium,washing with PBS once and then treating with TrypLE enzyme for 5minutes. After neutralization of TrypLE enzyme by adding equal volume ofmedium, cells were stained for flow cytometry. Briefly, cells werecentrifuged to remove culture medium and incubated with antibody for 30minutes at 4° C. Cells were then washed with wash buffer (PBS with 0.5%human serum albumin, 2 mM EDTA and 0.05% sodium azide) before use foranalysis. To assess hematopoietic cell generation, anti-CD38 PE,anti-CD41 FITC, anti-CD43 PE, anti-CD73 PE, and anti-hematopoieticprogenitor cell antibody PE (BD biosciences, San Jose, Calif.) wereused. Anti-CD14 FITC, anti-CD26 PE, anti-CD45 PE, anti-CD235a FITC(eBioscience, San Diego, Calif.), and anti-CD133 PE (Miltenyi Biotec,Auburn, Calif.) antibodies were also used for analysis.

Cell Isolation for Tissue Progenitor Cell Enrichment

Cells from ESC-MSC-Monocyte/Macrophage co-culture were sorted usingmagnetic bead-based separation to enrich tissue progenitor cells. ForCD34⁺ cell isolation, trypsinized cells were sorted by CD34 microbead(Miltenyi Biotec, Auburn, Calif.) in the presence of Fc receptor blocker(Miltenyi Biotec, Auburn, Calif.) to block non-specific interaction.

Real Time PCR Analysis

RNA from progenitor cells enriched by magnetic bead separation wasisolated with RNeasy mini kit (Qiagen, Valencia, Calif.) according tomanufacturer's instructions. RNA quality and concentration was assessedusing a NanoDrop spectrophotometer (Thermo Scientific, Waltham, Mass.).RNA was converted to cDNA using Quantitect Sensiscript kit (Qiagen,Valencia, Calif.). Power SYBR green master mix (Applied Biosystems) wasused for setting up RT-PCR plates according to the manufacturer'sprotocol. RT-PCR was performed using primers for GATA1, GATA2, GATA3,Flk1, Flt3, SCIL, and CD34 (Qiagen, Valencia, Calif.). Primers for 18SrRNA, GAPDH, and b-Actin (QIAGEN, Valencia, Calif.) were used ashousekeeping gene controls. StepOnePlus system (Applied Biosystems) wasused for detection.

Results

ESC-MSC-macrophage co-cultures generated CD34⁺ hematopoietic cells whenthe MSCs were derived from bone marrow (FIG. 2A). When MSCs were derivedfrom non-bone marrow sources, such as pancreas, co-cultures produced fewif any CD34⁺ cells (FIG. 2B). Co-cultures without MSCs or withoutmacrophages (FIG. 2C) failed to produce CD34⁺ cells, while small numbersof CD34⁺ cells were produced in the absence of CD14⁺ cells.

Flow cytometry using anti-CD14 antibodies confirmed that the CD34⁺staining was not due to non-specific antibody binding to Fc receptors.Co-staining with anti-CD73 antibodies confirmed that the CD34⁻ cells donot express CD73, a marker for MSCs. CD34⁺ cells also did not expressCD13, CD14, CD31, CD38, CD41, CD43, CD45, CD73, CD90, CD235a, and CD309(FIG. 9), suggesting that the cells are not endothelial cells, asindicated by the lack of CD31 expression, and are still uncommitted tospecific lineages. Some CD34⁺ cells generated in co-cultures expressedCD26 or anti-HPC antibody. The purity of the CD34⁺ cell population wasbetween 1 to 10%. Approximately 200,000 CD34⁺ cells could be generatedper well of a 6-well plate, a yield adequate to produce numbers of CD34⁺cells sufficient for clinical applications. Up to 20% ofnon-macrophage-, non-apoptotic cells expressed CD 34 after co-culture(FIG. 4). Hypoxia increased the yield of CD34⁺ cells after co-culture.

To confirm that the CD34⁺ cells were hematopoietic cells, CD34⁺ cellswere isolated using magnetic beads. Real time PCR on RNA from theisolated CD34⁺ cells revealed that these cells express GATA2, GATA3,Flk1, Flt3, and CD34. The CD34⁺ cells also expressed FOXA2. Whileco-cultures with non-bone marrow derived MSCs (e.g., pancreas MSCs)produced very few CD34⁺ cells, the few CD34⁺ cells derived from thosecultures had several fold lower expression of MSC, GATA3, Flt3, andCD34.

To rule out the possibility that the results of the real time PCRanalysis were due to contaminating MSCs, real time PCR was performed onbone marrow MSC and pancreatic MSC. The result shows that although bonemarrow MSCs express some of these genes, the level of expression forGATA3, FLT3, and CD34 was significantly lower compared to that of theCD34⁺ cells isolated from bone marrow-derived MSC co-cultures.

Example 3 Tissue-Specific Progenitor Cell Engraftment In Vivo

Cells

Cells were isolated and co-cultured essentially as described in Examples1 and 2. CD34⁺ cells were isolated from ESC-MSC-Monocyte/Macrophageco-cultures using magnetic bead-based separation to enrich tissueprogenitor cells.

Engraftment

To determine if the CD34⁺ cells isolated from MSC-pluripotent cell-CD14⁺cell co-cultures can be engrafted and, subsequently, reconstitute ahematopoietic system, the pre-immune fetal sheep model was used. Thefetal sheep model can be used to analyze multi-organ engraftment ofhuman cells that are introduced in-utero prior to the development of thefetal immune system. Successful engraftment within the resultinghuman/sheep hematopoietic chimeras can be analyzed after birth. Theengraftment was conducted essentially as described by Narayan et al.,Blood 107:2180 (2005), incorporated herein by reference as if set forthin its entirety. On gestation day 61, three animals were engrafted with0.5×10⁶ CD34⁺ cells each. The cells were delivered intra utero inserum-free media in a volume of 0.5 mL per fetus. After birth (gestationday 145-150), peripheral blood samples from engrafted and control lambswere analyzed using flow cytometry for the presence of human CD45⁺cells. The cells successfully engrafted and gave rise to 0.5-3.28% humanCD45⁺ cells in sheep peripheral blood (FIG. 3). This is a significantincrease over previous attempts of human cell engraftment that resultedin only 0.1-0.2% engraftment (Narayan et al.). These results indicatethat the methods described herein can be used to produce cells withsuperior ability to engraft into a recipient. As such, the methodsdescribed herein produce HSCs capable of long-term engraftment.

Example 4 Pancreatic Islet-Derived MSCs and Macrophages StimulateDifferentiation of hES Cells Into Pancreas Cells In Vitro

Cells

Monocytes were isolated essentially as described in Example 1. The ESCline WA01 was supplied from WiCell Institute and maintained on Matrigel™essentially as described in Example 2. Pancreatic islet MSCs werederived from pancreatic islets collected from cadaveric donors perintegrated islet distribution program protocol (IIDP) (available at theDiabetes Research Centers). Pancreatic islets were processed and yielded10,000 to 20,000 islet equivalents. Islet cells were cultured in humanMSC culture medium for several days until they attached to the cultureflasks. Human MSC culture medium contained αMEM medium supplemented with10% FBS. In some cases, non-essential amino acids and L-Glutamine wasalso added to the medium. Cells were harvested with TrypLE and passagedup to passage 4. Islet-derived MSCs were characterized by flow cytometryas described in Example 1 and were tested for differentiation potential.Briefly, pancreatic islet-derived MSCs were cultured in nonhematopoieticstem cell differentiation medium (Miltenyi Biotec, Auburn, Calif.) fordifferentiation into adipose, chondrogenic, and osteogenic tissueaccording to the manufacturer's protocol. The cells differentiated intocells of all three lineages, demonstrating that the cells were MSCs.

MSC-ESC Co-Culture

MSCs were plated into 6-well plates at a density of 1×10⁵-3×10⁵ cellsper well essentially as described in Example 2 except that forpancreatic islet-derived MSCs, C10 medium was used which was made bysupplementing CMRL1066 medium (Mediatech, Manassas, Va.) with 10% FBS(Atlanta Biologicals, Lawrenceville, Ga.), 1% NEAA, and 1%L-Alanine-L-Glutamine (Mediatech, Manassas, Va.).

MSC-ESC-CD14⁺ Cell Co-Culture

CD14⁺ cells were added to ESC-MSC co-cultures at a concentration ofabout 1×10⁶ cells per well essentially as described in Example 2. TheESC-MSC-CD14⁺ cell co-cultures were incubated in C10 media, which waschanged every three to four days by collecting floating cells in thesupernatant through centrifugation and resuspension in fresh mediumwhich was then added back to the original culture plates.

ESC-MSC-Macrophage Co-Culture Analysis Using Flow Cytometry

For flow cytometric analysis, cells were collected, washed, and stainedessentially as described in Example 2 except that CD14 FITC(eBioscience, San Diego), CD73 PE (BD Biosciences, San Jose, Calif.),HPi2, HPx2, HPd1, and HPa1 (Stemgent, Cambridge, Mass.) antibodies wereused. These antibodies recognize pancreas-specific cell markers that canbe used to enrich specific pancreas lineage cells. Dorrell, Stem CellRes. 1(3):183-194 (2008). Goat anti-mouse FITC secondary antibody orzenon Alexa 488 mouse IgG1 kit were used for signal detection.

Cell Isolation for Tissue Progenitor Cell Enrichment

Cells from ESC-MSC-CD14⁺ cell co-cultures were sorted using magneticbead-based separation to enrich tissue progenitor cells. Harvested cellswere depleted of macrophages using CD14 microbeads. Negative fractionsfrom CD14 isolation were incubated with HPi2 antibody for 15 minutes at4° C. and then washed with autoMACS running buffer. Cells were thenincubated for 15 minutes with anti-mouse IgG microbeads (MiltenyiBiotec, Auburn, Calif.) and subsequently positively selected by autoMACSpro system. Both HPi2 positive and negative fractions were kept forcomparison purposes.

Real Time PCR Analysis

RNA from progenitor cells enriched by magnetic bead separation wasisolated and subjected to real time PCR analysis essentially asdescribed in Example 2. RT-PCR was performed using primers for FOXA2,Insulin, Glucagon, PDX1, SLC2A2, G6PC2, and SOX7 (Qiagen, Valencia,Calif.). Primers for 18S rRNA, GAPDH, b-Actin (QIAGEN, Valencia, Calif.)were used as housekeeping gene controls. StepOnePlus system (AppliedBiosystems) was used for detection.

Results

ESC-MSC-macrophage co-cultures generated pancreatic islet progenitorcells when the MSCs were derived from pancreatic islets. Severalmonoclonal antibodies known to enrich certain populations of pancreaticcells were used to detect pancreatic islet progenitor cells. HPi2antibody which is known to enrich islet cells stained the resultingprogenitors. When the MSCs used for co-culture were derived from bonemarrow, few cells stained with HPi2 antibody. The pancreatic islet cellsgenerated in co-culture did not stain with HPx2 (exocrine cells), HPa1(pancreatic alpha cell), or HPd1 (pancreatic duct) antibodies. Thesecells also did not express CD14 and CD73, confirming that these cellswere neither macrophages nor mesenchymal cells.

Example 5 In Vitro Progenitor Development Testing Platform

The co-culture system described in Example 2 was used as an in vitrotesting platform to determine the potential effect of agents ontissue/organ development. Cells for ESC-MSC-CD14⁺ cell co-cultures wereprepared essentially as described in Example 1. A non-specific Wntpathway agonist (cat. No. 681665-5 mg, EMD Millipore, USA) was testedfor its effect on CD34⁺ cell generation as Wnt1 activation is known toplay role in hematopoiesis during embryonic development. Significantlyfewer CD34⁺ cells were present in co-cultures in the presence of the Wntpathway agonist, compared to co-cultures without the agonist (FIG. 5).This result suggests that the Wnt pathway agonist might interfere withhematopoiesis.

Example 6 MSCs-Macrophages Co-Culture Stimulates Proliferation of CD34⁺Cells In Vitro

MSCs were harvested from healthy BM and passaged as described inExample 1. Macrophages were derived as described in Example 1.MSCs-macrophage co-culture experiments were conducted as described inExample 2. Human G-CSF mobilized CD34⁺ cells were stained with 10 μMcarboxyfluorescein diacetate succinimidyl ester (CFSE) for 15 minutes at37° C., washed, and seeded into 6-well plates with a) no supportivecells, b) MSCs, c) macrophages, or d) MSCs and macrophages. CFSE is adye that penetrates cell membranes and becomes trapped inside cellsafter intra-cellular enzymatic cleavage. The amount of CFSE per celldecreases by half with each cell division. Cells were cultured for fourdays and subsequently analyzed by Fluorescence-activated cell sorting(FACS) and proliferation indices were calculated using ModFit software.The proliferation index (PI) is the sum of the cells in all generationsdivided by the calculated number of cells initially subjected to theassay. CD34⁺ cells co-cultured with MSCs or macrophages showed higherlevels of proliferation (PI=2.09 and PI=1.82, respectively) compared toCD34⁺ cells cultured in the absence of these cells (PI=1.31). CD34⁺cells co-cultured with both MSCs and macrophages show the highest rateof proliferation (PI=2.80). This data suggest that MSC-macrophageinteractions support CD34⁺ cell proliferation, a contribution ofmacrophages not previously investigated.

Example 7 In Vitro Expansion of Multiple Myeloma Cells

The MM cell lines U266 and NCI-H929 were acquired from American TypeCulture Collection (ATCC) and were cultured according to recommendedprotocol. Briefly, U266 cells were cultured in RPMI 1640 with 10% FBS,1% NEAA, 2 μM L-alanine-L-glutamine and 1% sodium pyruvate, and mediumwas changed every two to three days. U266 MM cells were stained with 10μM CFSE for 15 min at 37 C, washed twice in PBS containing 0.5% HSA, andadded to 6-well plates at a concentration of 200,000 cells per well witha) no supportive cells, b) MSCs, c) macrophages, or d) MSCs andmacrophages. Cells were cultured for four days and subsequently analyzedby flow cytometry. Proper gating was necessary as some macrophages weredimly positive for CFSE because they had phagocytosed CFSE-stained MMcells. Proliferation indices were calculated with ModFit software.

FIG. 6 depicts the mean proliferation index (PI) calculated from threeseparate experiments, each performed in triplicate, using MSCs andmacrophages (MQ) from separate healthy donors. MSCs enhanced U266proliferation (PI=7.24) compared to U266 grown in the absence ofsupporting cells (PI=4.21). This is consistent with the long-held beliefthat MSCs act as a main stromal component for MM malignancy. Hideshimaet al., Nat. Rev. Cancer 7:585-598 (2007). Interestingly, macrophagesenhanced U266 proliferation (PI=11.84) beyond that observed with MSCs.The highest level of U266 proliferation could be observed in culturescontaining both MSCs and macrophages (PI=12.92). Similar results wereconsistently observed in further experiments using both U266 or NCI-H292MM cell lines. Similar results were also obtained when the experimentswere conducted using RPMI1640 with 10% FBS and RPMI1640 with 0.5% humanserum albumin, demonstrating that the observed increase in MM cellproliferation was not due to contaminating macrophage media (IMDMsupplemented with 10% human serum AB). These results suggest that themacrophage-MSC interaction enhances tumor cell growth.

Example 8 MSC-CD14⁺ Cell Co-Culture With Normal or Malignant CellsReduces Apoptosis In Vitro

In vitro maintenance of primary malignant cells is challenging in partbecause the cells undergo programmed cell death (apoptosis). Todetermine if co-culture with MSCs and CD14⁺ cells affects apoptosis ofmalignant cells in culture, primary chronic lymphocytic leukemia cellswere cultured either alone (FIG. 7, CLL alone), with bone marrow MSCs(FIG. 7 MSC), with macrophages (FIG. 7, MQ), or with MSCs andmacrophages (FIG. 7, MSC+MQ). The cells were analyzed for Annexin Vbinding to the cell surface, a marker for early stage apoptosis, and forpropidium iodide staining, a marker for late stage apoptosis ornecrosis. Early apoptotic cells are Annexin V⁺/PI⁻, late apoptotic cellsare Annexin⁺/PI⁺.

Primary CLL cells exhibited decreased apoptosis in the presence ofeither MSCs or macrophages (FIG. 7). The greatest decrease in apoptosiswas observed when the primary CLL cells were co-cultured with both MSCsand macrophages (FIG. 7). Similar protection from apoptosis was observedfor primary B cells cultured with either macrophages or MSCs andmacrophages (FIG. 8), suggesting that the anti-apoptotic effect of MSCand macrophage co-culture is not limited to malignant cells.

We claim:
 1. A method for generating a tissue-specific progenitor, themethod comprising the steps of: co-culturing under xeno-free conditionsa peripheral blood CD14⁺ monocyte or a peripheral blood CD14⁺monocyte-derived macrophage, a mesenchymal stem cell (MSC) obtained froma tissue of interest, and a human pluripotent cell in vitro until thehuman pluripotent cell differentiates into a cell exhibiting a phenotypecharacteristic of a progenitor of a cell of the tissue of interest,wherein the human pluripotent cell is selected from the group consistingof a human embryonic stem cell and a human induced pluripotent stemcell, and wherein when the tissue-specific progenitor is implanted invivo into a host subject it differentiates into a cell of the tissue ofinterest.
 2. The method of claim 1, wherein the CD14⁺ cell is amonocyte.
 3. The method of claim 1, wherein the peripheral blood CD14⁺monocyte-derived macrophage is obtained by culturing under xeno-freeconditions a human peripheral blood monocyte until the peripheral bloodmonocyte exhibits a phenotype characteristic of a CD14⁺ monocyte-derivedmacrophage.
 4. The method of claim 1, wherein the tissue-specificprogenitor is selected from the group consisting of a hematopoieticprogenitor, a cardiomyocyte progenitor, and a pancreatic isletprogenitor.
 5. The method of claim 1, wherein the tissue of interest isselected from the group consisting of bone marrow, heart, pancreas,lung, liver, skin, and kidney.